Jackie Krevitz Sordaria Lab Report Nathan Jacobs October 29, 2012

Introduction: Sordaria fimicola is a species of fungi that can reproduce both sexually and asexually and is used in research to learn more about microevolution1. Sordaria is an ideal specimen to study because of its short reproduction time, the ability to manipulate crosses, and the fact that it produces asci that are easily viewed under a microscope1. When Sordaria sexually reproduces, it obtains genetic material from each parent. During meiosis, crossing over can occur between the two strains of Sordaria, which results in recombination1. Sordaria produce perithecia, fruiting bodies, which contain asci1. The asci contain eight ascospores, which allow researchers to calculate the recombination frequency of the Sordaria1. When a Sordaria sexually reproduces with a strain that is the same color as itself, all eight of the ascospores within the asci are the same color1. When Sordaria sexually reproduces with a strain that is a different color than itself, but no recombination occurs, the ascospores appear in a 4:4 ratio, with four of one color (in our case black) and four of the other color (tan) 1. If recombination occurs during meiosis, the ascospores appear in either a 2:2:2:2 ratio, black:tan:black:tan and tan:black:tan:black, or 2:4:2 ratio, black:tan:black and tan:black:tan2. By observing the many asci of one perithecia, the recombination frequency overall can be calculated, and therefore the distance from the color gene to the centromere of the Sordaria DNA can be calculated as well1.

An Evolution Canyon is a site where a barrier divides a species into two or more groups2. Because of the barrier, the two groups cannot mate and their gene pools become separated, and therefore one can see microevolution occur. One example of an evolution canyon is in Israel, located in Lower Nahal Oren, Mount Carmel2. One side of the canyon receives more sunlight, has a higher overall temperature, and receives less rainfall2. The other side is cooler, has more shade, and receives more rainfall2. Researchers collected Sordaria samples from each side of the canyon, and created wild type strains of each Sordaria strain in the laboratory2. They crossed the two strains as well, and calculated recombination frequency2. What the researchers found was that recombination frequency increases under environmental stress2. A factor contributing to this could be that there are advantages and disadvantages to sexual and asexual reproduction2. Sordaria have the ability to reproduce both sexually and asexually1. Asexual reproduction is beneficial in that it occurs quickly and uses less energy1. However, asexual reproduction reduces the genetic variability in a gene pool, because it only produces clones of the parent species. Sexual reproduction takes more time and energy, yet in introduces genetic variability into a gene pool1. If something occurs that makes a certain gene very harmful to a species, and there is not a lot of genetic variability, that species could become extinct. Genetic variability helps a species survive in a changing environment. This helps explain why environmental stress causes recombination frequencies to increase2. If the Sordaria sense that the environment is changing, it will sexually reproduce in order to increase the genetic variability and give the species a better chance at survival2. Some research questions that come out of this background research are as follows. The purpose of this experiment is to determine if crossover occurs during the color mutant

and wild type crosses. As well, the objective is to see what the overall crossover frequency of each of the color genes with the centromere in organisms. Another goal is to find out the frequencies of the two crossover types for each color strain, and to find any variation in the ratio of the two crossover types between the two color strains. In our experiments, we crossed two strains of Sordaria fimicola, wild type x tan and wild type x gray. We calculated recombination frequencies and the distance to the centromere of the color gene.

Hypothesis: Crossover will occur during the color mutant (tan or gray) and wild type (black) crosses. Based on research, the crossover frequency will be around 50%, and based on that, the distance between the centromere and the color gene in Sordaria will be around 25. The frequencies will not differ greatly between the tan and gray strands of Sordaria, because although we are using different strands, everything is still part of the same species. The genes are located in roughly the same places.

Materials and Methods The agar plate was divided into four quadrants. Two .5 cm by .5 cm black Sordaria squares were cut from the black Sordaria tray using a scalpel and placed into diagonal quadrants on the agar plate. Then, two squares of tan Sordaria (the lab calls for both tan and gray, but our group did just tan) were cut from the tan Sordaria tray and placed onto the two remaining quadrants of agar. Two weeks were allowed for full growth of the Sordaria. Using an inoculating loop, Sordaria was gathered from the middle of the agar

plate where the black and tan strains intersected. This sample was placed onto a slide with a drop of water and a cover slip, then gently squashed to break the asci out of the perithecia so they could be viewed under a microscope. The different patterns of asci were counted and recorded. Our groups specimens were premature, and the colors of the asci could not be seen. So, we used a pre-prepared slide and counted the asci patters. The recombination frequencies were calculated.

Data: Table 1: Individual Data NonRecombinant # of Type A Asci 7 Recombinant # of Type B Asci 7 # of Type C Asci 6 Total # of Asci 20 Total # Recombinant Asci 13

Table 2: Combined Lab Group Data NonRecombinant Recombinant # of Type A # of Type B # of Type C Asci Asci Asci 17 12 11

Total # of Asci 40

Total # Recombinant Asci 23

Table 3: Combined Section Data NonRecombinant Recombinant Total # of Asci # of Type A # of Type B # of Type C Asci Asci Asci Tan Spore Color 51 35 47 18 35 Gray Spore Color 26 79 133

Total # Recombinant Asci 82 44

Table 4: Combined Course Data NonRecombinant # of Type A Asci 5669 3012 Recombinant # of Type B Asci 4301 2081 # of Type C Asci Tan Spore Color 3976 Gray Spore Color 1973 7066 4054 Total # of Asci Total # Recombinant Asci 8277

13946

Table 5: Combined Section Data Analysis NonRecombinant Recombinant Total Total # Frequency of # of Recombinant Recombinant # of # of # of Type A Asci Asci Type Type C Asci Asci B Asci Asci Tan Spore Color 51 47 35 133 82 Gray Spore Color 35 18 26 79 44 0.556 0.616

Frequency of Type B Asci

Frequency of Type C Asci

Ratio B/C

0.353

0.263

1.34

0.227

0.329

0.692

Table 6: Combined Course Data Analysis NonRecombinant # of Type A Asci Recombinant # of Type B Asci 4301 # of Type C Asci 3976 Total # of Asci Total # Recombinant Asci Frequency of Recombinant Asci Frequency of Type B Asci Frequency of Type C Asci Ratio B/C

Tan Spore Color 5669

13946

8277 Gray Spore Color

0.593

0.308

0.285

1.08

3012

2081

1973

7066

4054

0.574

0.294

0.279

1.05

According to the course data, the distance from the centromere for the tan spore color is 29.7 mu. The gray spore color is 28.7 mu from the centromere. I calculated this using this course data because there was the most data, and therefore the results would be the more accurate.

Discussion: The crossover frequency between gray and tan spore crossed to the black wild type spore is almost identical. The tan spore color hovered around 55-60% recombination. Based on this recombination frequency, the map distance to the centromere is 29.7 mu and 28.7 mu for tan and gray spore colors, respectively. From the course data, I noticed a slightly higher recombination frequency of the type B than the type C asci for both tan and gray spore colors. According to other published studies, the accepted value for the spore color gene in Sordaria fimicola is 26 mu3. Both of my values are close to this accepted value, but not quite there. The percent error for gray is 14.2% and the percent error for tan is 10.4%. An unexpected finding was that there were usually more type B recombinant asci than type C recombinant asci. I would expect it to me more equal. In the evolution canyon, a higher mutation rate was found in the south-facing slope (more sunlight, less water) than on the north facing slopes (more shade, more humidity). They found that there were big differences in crossover frequencies between the two slopes, which helps support the claim that harsh environmental conditions cause an increase in recombination2. One implication of the findings from this experiment in regards to the research question about environmental stress and crossover frequency in Sordaria is more support for the theory of evolution. If Sordaria fimicola can adapt in regards to microevolution to its surroundings, this gives support for evolution as a whole. One possible source of error in this experiment was that many of our spores did not have enough time to fully mature. This made it very difficult to determine the ascospore colors, and therefore the recombination frequencies of the Sordaria. Another source of

error could have been that due to us squashing the perithecia, some of the asci were split apart, and the ascospores were rearranged a bit. This caused us to not be able to tell which type (A, B, or C) the asci were. Another source of error was human error, but the large sample size of the all course data helps reduce or eliminate human error in our calculations. A new research question that relates to this research is what is the effect of UV radiation (e.g. from sunlight) on the recombination frequency of gray x black and tan x black Sordaria fimicola. Is there a significant difference between the frequencies in the two crosses? UV radiation could be considered an environmental stress, which would hypothetically trigger an increase in recombination. As well, UV radiation causes DNA mutations, and it would be interesting to see what effect this would have in further experimentation.

Works Cited 1. Kalogeropoulos, A., & Thuriaux, P. (1985). Gene Conversion at the Gray Locus of Sordaria Fimicola: Fit of the Experimental Data to a Hybrid DNA Model of Recombination. Genetics Society of America, 599-610. Retrieved from PubMed database. 2. Lamb, B.C., Mandaokar, S., BAhsoun, B., Grishkan, I., & Nevo, E., (2008). Differences in spontaneous mutation frequencies as a function of environmental stress in soil fungi at Evolution Canyon, Israel. Proceedings of the National Academy of Science, 105(15), 57925796. 3. Olive L. (1956) Genetics of Sordaria Fimicola. I. Ascospore Color Mutants. American Journal of Botany, 43(2), 97-107.